After preparing HRP and storing it in a refrigerator at 4 degrees Celsius, how long can the activity generally remain stable without decreasing?

How long can HRP be stored at 4 degrees Celsius after opening for commercialization? The stability given by different manufacturers varies, and it is generally safer to use it up within 1-3 months after opening. The key is sterile use, and the gun should not touch the bottle mouth to avoid microbial degradation of enzymes. If you purchase a ready to use HRP secondary antibody containing stabilizers/preservatives, store it according to the instructions. After opening, it can usually be stored for several months at 4 degrees Celsius, but it is not recommended to store it for a long time after diluting the working solution. It is recommended to pack the goods into brown tubes according to each usage after arrival, avoiding light at 4 degrees Celsius. Take one tube and one tube, and record the opening date and activity control.

Glycerol is mainly used for -20°C antifreeze. At a final concentration of 30%-50%, HRP stock solutions do not freeze at -20°C and can be taken out for direct use, which is gentler than repeated freeze-thaw cycles. At 4°C, glycerol cannot replace preservatives; it is still recommended to add ProClin or thimerosal. I once prepared an HRP stock solution with 50% glycerol + 0.02% ProClin and stored it at -20°C, and WB color development remained stable within six months. At 4°C, if a preservative is added, activity is usually acceptable within 1-2 months, but it is recommended to perform a substrate color development control every two weeks. Note that glycerol changes the viscosity of the solution, so thorough mixing is required when diluting for IHC.

Rather than worrying about how long it can be stored at 4°C, it is better to aliquot the stock solution. A 1 mg/mL stock solution can be aliquoted at 20-50 μL/tube and stored at -20°C to avoid repeated freeze-thaw cycles; if used in large amounts, add 30%-50% glycerol and store at -20°C, so that repeated freeze-thaw cycles are avoided during retrieval. Inactivation caused by freeze-thaw is often more pronounced than short-term storage at 4°C. You mentioned that both WB and IHC are used, so it is recommended to prepare two portions: one containing glycerol for long-term storage at -20°C, and one working solution at 4°C to be used up within one week. This avoids daily freeze-thaw cycles and also ensures activity. If the color development becomes lighter or the background is abnormal, first suspect the shelf life of the stock solution.

HRP storage solution is stored at 4 degrees away from light, and in experience, it can remain stable for 2-4 weeks in PBS containing protective proteins, with no significant decrease in activity; I generally do not recommend using it for critical experiments after 4 weeks. The recommended buffer solution is pH 7.2-7.4, with 0.1% BSA added as a protective protein. Do not use sodium azide for preservation, as it will inhibit HRP. If it is necessary to store at 4 degrees for a longer period of time, 0.02% ProClin 300 or 0.01% Thimerosal can be added, but it is best to verify the activity with a small sample. Frequent use of secondary antibodies is recommended to be divided into weekly doses to reduce repeated lid opening and contamination.